Effect of Telmisartan on Glucose Homeostasis of Skeletal Muscle and Its Mechanism

Background and Purpose: The metabolic syndrome, which describes the presence of visceral obesity, insulin resistance or glucose intolerance, atherogenic dyslipidemia, hyperuricemia, and hypertension in a patient, increases the risk for type 2 diabetes and cardiovascular disease. A class of anti hypertensive drugs, angiotensin II type 1 receptor blockers (ARBs), has been reported to have favorable effects on insulin sensitivity. Particularly, sub analysis of several recent clinical trials has revealed that these drugs reduce the incidence of new onset diabetes. Additionally, blockade of the AT1R has been shown to improve insulin sensitivity in animal models of insulin resistance. However, the mechanisms underlying the insulin sensitizing and antidiabetic effects of the ARBs have not been defined. They may include enhancing blood flow through the microcirculation of skeletal muscles and, thereby, promotion insulin and glucose delivery to the insulin sensitive tissues. As a result, insulin signaling is facilitated at the cellular level and insulin secretion by beta cells is improved. Some ARBs, such as, telmisartan (Tel), have been found to effectively activate the peroxisome proliferator activated receptorγ(PPARγ) , a well known target for insulin sensitizing.PPARs are ligand – inducible transcription factors which isoforms display tissue specific expression and gene regulatory profiles. PPARγis a key regulator of adipocyte differentiation and adipose insulin sensitivity, whereas it is barely expressed in skeletal muscle. By contrast, PPARδ(also referred to as PPARβ) is expressed in a wide variety of tissues, with high levels in skeletal muscle. Studies in transgenic mice show that targeted expression of activated PPARδincreases the predominance of oxidative typeⅠmuscle fibers, enhances whole body insulin sensitivity, and increases exercise endurance capacity . The PPARδspecific agonist GW501516 improves insulin sensitivity in skeletal muscle in several animal models. Thus, activation of PPARδmay offer an efficacious strategy for the improving glucose homeostasis. However, this particular PPARδagonist is not available for clinical use due its undesired side effects. It is important to know whether ARBs, such as Tel affect PPARδexpression and/or function. Given the importance of skeletal muscle insulin resistance in the development of type 2 diabetes, we hypothesized that Tel has direct effects on glucose metabolism in skeletal muscle through an action on PPARδ.In the present study, we first confirmed whether Tel directly improved glucose intake in primary cultured myotubes of mice throught PPARδ. Then we detected the glucose metabolism related key molecules in skeletal muscle cell and tissue of mice to find out whether PPARδwere regulated by Tel. Finally, we examined the glucose metabolism related struction and function changes of skeletal muscle in C57BL/6J and PPARδknockout mice fed with or without Tel.Methods:The present study includes in vivo and in vitro experiments. In vitro models were primary cultured myotubes from C57BL/6J (C57)and PPARδknockout mice, as well as myotubes differentiated from control C2C12 myoblasts. In vivo models include wild type mice and PPARδknockout mice fed with or without telmisartan, and spontaneous hypertensive rats (SHR) were also observed.1. The differentiated C2C12 myotubes were divided into groups as follow: Control, Insulin, Insulin+angtensionⅡ(AngⅡ)and Insulin+ AngⅡ+Tel groups. After treatment, the 2 deoxyglucose (2 DG) uptake was assayed.2. The skeletal muscle cells from C57 and PPARδknockout mice were cultured in DMEM using the primary culture technique. After confluent, cells were given DMEMs without FBS. Telmisartan was added to media in present or absence of Compound C. After 24 h, 2 Deoxyglucose uptakes assay in cells were carried out.3. Protein expression of glucose metabolism related molecules including PPARδ, PPARγ, AMPKα1, p AMPKα, TroponinⅠss as well as RAS components (included ACE、AngⅡ、Renin and AT1R) in skeletal muscle cells and tissue from C57 and PPARδknockout mice, were detected by immunoblotting and mRNA expression of PPARδand PPARγwere detected by RT PCR. Protein and mRNA expression of PPARδand PPARγin SHR were also analyized.4. Mice were fed a normal diet or normal diet plus telmisartan. Daily food intake per mouse was recorded during the ?rst 10 days after the start of telmisartan administration. Body weights were measured every two weeks throughout the experimental period. During 23 24 weeks, exercise endurance, oxygen consumption, intraperitoneal glucose tolerance test (IPGTT) as well as insulin tolerance test (ITT) were analyzed in mice. After sacrificed, metabolic parameters were determined and fiber typing of gastronomies muscle was observed using the metachromatic dye ATPase method.Major Research Results:1. The 2 DG uptake in differentiated C2C12 myotubes increased by insulin and inhibited by AngⅡprincubation. Telmisartan inverted the inhibition effect of AngⅡon the insulin stimulated 2 DOG uptake .2. Insulin and telmisartan significantly increased 2 DG uptake in cultured myotubes from C57 mice compared with control myotubes. Administration of compound C, an AMPK inhibitor, significantly reduced telmisartan’s action in cultured C57 myotubes. In the cultured PPARδdeficient myotubes, insulin increased 2 DG uptake, but telmisartan no effect on glucose uptake.3. Telmisartan significantly increased expression of PPARδmRNA and protein expression in cultured myotubes in skeletal muscle tussue from C57 mice, and no effect on PPARγexpressions in skeletal muscle cell and tissue from either C57 or PPARδdeficient mice. Protein expression of AMPKα1and p AMPKαin myotubes , p AMPKαand TroponinⅠss in skeletal muscle tussue of C57 was significantly increased by telmisartan stimulation. Telmisartan did not influence RAS components of primary cultured myotubes and skeletal muscle in either wild type or PPARδdeficient mice.4. 24 week telmisartan treatment reduced weight gain in C57 mice, but not in PPARδdeficient mice. Food and water intake was not affected in either group. Serum insulin and triglyceride levels did not differ between mouse strains with or without telmisartan treatment, but cholesterol lower in telmisartan treatment group of C57 micel. Compared with control mice, C57 mice treated with telmisartan had higher exercise endurance, post exercise oxygen consumption andⅠskeletal muscle fiber composition, lower AUC of IPGTT and ITT, as well as lower blood glucose level after insulin injection in ITT. However, PPARδdeficient mice treated with telmisartan displayed no phenotypic change relative to untreated PPARδdeficient mice. Conclusion1. Telmisartan directly increases glucose uptake in skeletal muscle cell through PPARδ/AMPK path. When RAS actitivity enhancement, telmisartan improves the insulin stimulated 2 DOG uptake by inhibition effect of AngⅡon skeletal muscle cell.2. Through activating PPARδ/AMPK path, telmisartan reduces weight gain, increasesⅠskeletal muscle fiber composition in gastronomies muscle and post exercise oxygen consumption, enhances exercise endurance, improves glucose tolerance and increases insulin sensitivity in mice.3. Telmisartan has no effect on RAS components in myotubes and skeletal muscle tissue of mice in normal condition.